PROJECT SUMMARY Despite having a dedicated copper export system, copper has been broadly toxic to bacteria since antiquity. Copper toxicity is seen in practice where copper surfaces and tools significantly reduce nosocomial infections. Furthermore, during host mediated nutritional immunity (sequestering essential metals while bombarding bacterial with toxic metals), innate immune cells kill engulfed targets using copper, which is tightly regulated within the host. However, current research is limited regarding the overarching mechanisms of toxicity and the pathways used overcome copper stress that work in conjunction with the copper export system. Through this proposal, we seek to understand how bacteria evolved to interact with toxic metals and overcome the ensuing stress. Furthermore, we seek to gather a new cohort of bacterial therapeutic targets. For our model system, we are using Streptococcus pneumoniae, a global burden, for which the vaccine only covers ~25% of known strains. Thus far, using microarray data we obtained comparing the wild type S. pneumoniae strain TIGR4 and the copper export protein deficient strain under copper stress, and an assortment of techniques, we have validated several pathways and their corresponding mechanisms. These pathways include copper poisoning nucleotide synthesis, zinc being synergistically toxic to bacteria with copper, and sugar import being vital to mediation of copper toxicity. We will focus our studies around the copper repressor protein CopY as we have data showing it not only controls the copper export system but several other systems necessary to overcome copper stress. We will characterize these CopY-controlled pathways as well as structurally characterize CopY, screen CopY for small molecule therapeutics that keep it bound to DNA, and examine CopY?s interactions with DNA and proteins. We will expound upon our current findings, explore new avenues of how copper affects S. pneumoniae, and apply our findings to other toxic metals and bacteria in hopes to elucidate the orchestrated bacterial response to metal stress.